Method and device to probe a membrane by applying an in-plane electric field

ABSTRACT

The present invention disposes a membrane between two electrical conductive walls having a height at least as great as the thickness of the membrane. The conductive walls are fabricated on an electrically insulative chip base. The chip base has one or more through hole between the electrically conducting walls. The chip is placed inside a container having a well below the through hole of the electrically insulative base. At least one passageway extends from the well to the periphery of the container. This invention probes changes of the membrane as an in-plane electric field is applied between the conductive walls. The well may include various compounds while other compounds can be placed in contact with the top of the membrane. The passageways are used to introduce substances into and out of the well.

CROSS-REFERENCE TO RELATED APPLICATION

This application has the same inventorship as U.S. patent applicationSer. No. 11/400,685, filed on Apr. 7, 2006, entitled “Method and Devicefor Probing Changes in a Membrane by Applying an In-Plane ElectricField,” which is incorporated herein by reference in its entirety. Thisapplication also claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/824,817, filed Sep. 7, 2006.

BACKGROUND OF THE INVENTION

The membrane of the biological cell, consisting of amphiphilicglycolipids, phospholipids, cholesterol and proteins, is the outermostboundary that separates the intracellular components from theextracellular environment and is involved in a wide variety ofbiological processes. It is semipermeable and capable of regulating whatenters and exits the cell. The transport of substances in and out of thecell can take place with or without active participation of the cellmembrane. The surface of the cell membrane anchors the cytoskeleton andthe other molecules that activate or deactivate certain cell processes.Proteins embedded in the membrane act as selective channels for ions,receptors for information exchange between cells and organelles, andtake part in activities such as immune response and cell adhesion. Themembrane and the proteins carry out these functions mainly by changingtheir structure reversibly. How these structural changes take place andthe molecular mechanisms behind them are at the forefront of lifescience research.

The structure of the generally accepted fluid mosaic model of themembrane is a self assembling two dimensional smectic liquid crystallineamphiphilic lipid bilayer in which hydrophobic hydrocarbon chains areinside and hydrophilic polar headgroups are outside. However, recentstudies show that cell membranes contain different structures or domainsthat can be classified as protein-protein complexes; lipid rafts,pickets and fences formed by the actin-based cytoskeleton; and otherlarge stable structures, such as synapses or desmosomes.

The phase behavior of lipids in the membrane is known to be involved incell fusion processes and membrane traffic, for example, duringexocytosis or virus-cell fusion in the course of an infection. Thepropagation of action potential in nerve and muscle cell and retinalphotoreceptors have been attributed to the ferroelectric propertiesarising from chiral building blocks. A Curie point and current-voltagehysteresis typical of ferroelectric substances have been observed incell membranes. Temperature dependent current has been induced by laserin frog of ranvier suggesting a pyroelectric effect. Swelling ofmembranes in response to a voltage application, which indicates apiezoelectric effect, has been reported. It has been suggested thatferroelectricity may be common in cell components and a relationshipbetween liquid crystalline ferroelectricity and nerve and muscleimpulses has been predicted, but so far the possible origin of theferroelectric structure in the cell membrane has not been demonstrated.

Both glycolipids and phospholipids contain polar (hydrophilic) headgroups and apolar (hydrophobic) alkyl chains. They are quite similar intheir molecular shape and phase behavior. They are amphotropic liquidcrystals: they form thermotropic liquid crystalline phases in their pureform as the temperature is varied and lyotropic liquid crystallinephases in solvent as the concentration is varied. The length of thealkyl chain and the number of head groups determine the polymorphism inboth thermotropic and lyotropic structures. They form smectic bilayersin water at a critical concentration of lipids. Lyotropic properties ofboth glycolipids and phospholipids have been extensively studied in thelast decade but so far their thermotropic properties have not beenassessed properly. The thermotropic form of membrane lipids, bothphospholipids and glycolipids, presents a unique opportunity toinvestigate many of their physical especially electrical propertieswhich are more difficult to study in aqueous systems. Most membranelipids with two long alkyl chains form only a columnar phase in theirpure form. A smectic phase of these membrane lipids can be induced bymixing them with amphiphilic lipids which form only a smectic phase,providing ideal systems to investigate structural and electricalproperties of lipid bilayers.

Recently it was shown based on dielectric and X-ray diffraction studies,and optical microscopic observations that the glycolipid molecules aretilted in their bilayers in the smectic phase but the direction of thetilt is varied from one bilayer to the next. Large numbers of studiedsynthetic glycolipids with varying chemical structures exhibited quitesimilar behavior. The tilted supramolecular structures they from in bothbent-core and straight-core liquid crystals also show that lipidmolecules are tilted in the bilayers. The bilayers of tilted chiralglycolipid molecules are electrically polarized. It is also known thatamphiphilic lipids form only one smectic phase in both thermotropic andlyotropic form. Since the smectic phase in thermotropic form of theamphiphilic lipids is similar or identical to their smectic phase in thelyotropic form, the amphiphilic lipid bilayers may be polarized even inthe aqueous medium. Therefore, it is possible that the tilted lipidswill give rise to ferroelectric domains in the biological cell membranesas well. As a result of in-plane anisotropy and ferroelectricity of themembrane, the lipid bilayer may play an active role in determining theexcitable properties of the cell membrane.

Most proteins fold into unique three dimensional structures. The shapeinto which a protein naturally folds is known as its native state. Thereare four main protein structures known as primary, secondary, tertiary,and quaternary structures. In addition to the biochemical role of thesemain structures, proteins may shift between several related structuresin performing their biological function. In the context of thesefunctional rearrangements, these tertiary or quaternary structures areusually referred to as “conformations,” and transitions between them arecalled conformational changes. For example, the binding of a substratemolecule to an enzyme results in such conformational changes in physicalregions of the protein that participate in chemical catalysis.Discovering the tertiary and quaternary structure of protein complexes,can provide important clues about how the protein performs its function.

The main experimental methods of structure determination are X-raycrystallography and NMR spectroscopy, both of which can produceinformation at atomic resolution. At lower resolution, the cryo-electronmicroscopy is used to determine secondary structures of very largeprotein complexes such as virus coat proteins and amyloid fibers. Avariant known as electron crystallography is also used inhigh-resolution studies in some cases, especially for two-dimensionalcrystals of membrane proteins. Solved structures are usually stored inthe Protein Data Bank (PDB), a freely available resource from whichstructural data about thousands of proteins can be obtained in the formof Cartesian coordinates for each atom in the protein.

There are many more known gene sequences than there are solved proteinstructures. Further, the set of solved structures is biased toward thoseproteins that can be easily subjected to the experimental conditionsrequired by one of the major structure determination methods. Inparticular, globular proteins are comparatively easy to crystallize inpreparation for X-ray crystallography, which remains the oldest and mostcommon structure determination technique. Membrane proteins, bycontrast, are difficult to crystallize and are underrepresented in thePDB. Structural genomics initiatives have attempted to remedy thesedeficiencies by systematically solving representative structures ofmajor fold classes. Protein structure prediction methods attempt toprovide a means of generating a plausible structure for a protein whosestructures have not been experimentally determined.

DISCLOSURE OF THE INVENTION

It is an object of the invention to provide a method to probe dynamicconformational changes and associated functions in the biological cellmembrane and membrane proteins by applying an in-plane transverseelectric field. As this method is not dictated by purification andre-crystallization of the proteins, the proteins can be probed in theirnative environment. The membrane can be extracted from a biological cellor can be formed synthetically by individual constituents of extractedfrom the biological cell membrane or by synthetic molecules. Theproteins can be inserted into the membrane during and after theconstruction of the membrane. Subsequently, the membrane is subjected tothe in-plane electric field and probing its structural changes coupledwith the in-plane electric field.

It is a further object of the invention to provide a device with whichthe inventive method can be applied. The principle underlying both themethod and the inventive device is based on the insight that when analternating electric field is applied along the plane of the membrane,the in-plane electric field causes a structural transition in theconstituent molecules of the membrane. Membrane proteins acquire uniquestructures in response to the strength and the frequency of the electricfield and time evolution of these structural changes are observable inthe extracellular part of the proteins. In addition, structural changestaking place in the intracellural part of the proteins can be determinedby electrical characterizations. These dynamic structural changes may berelated to the biological functions of the membrane. Time evolution ofstructural changes cannot be probed by X-ray or NMR measurements in realtime. These molecular activities of the membrane now can be probed withthe present invention in that the structural changes can be induced andcontrolled by an external electric field and probed using a number ofmicroscopic and electrical measurements.

This invention relates to probing of the structural transitions of themembrane and the membrane proteins using electric current measurements,impedance gain phase analysis, raster scanning by atomic forcemicroscope, and further characterization of the membrane with confocalmicroscopy, X-ray spectroscopy and NMR, but not limited to these tools.

This invention applies to all aspects of membranes and, in particular,to the structural functions of biological cell membranes including thebiological sciences of the biological cell membrane, medical andclinical research of the biological cell membrane and diagnosis ofdiseases related to membrane and protein dysfunctions.

According to the invention, to apply an electric field in the plane ofthe membrane, the membrane is disposed between two electrodes forming acontainment region of a nanometer scale parallel plate capacitor. Oneside of each electrode is, for example, about 5-10 nanometers (1×10⁻⁹ mor nm) high so that it is not less than the height of the membrane and,in particular, is approximately the same height as the membrane. Thedistance between the electrodes is, for example, about 3-10 micrometers(1×10⁻⁶ m or μm) which is suitable for a sufficiently large electricfield. To enable access for probing, the membrane is positioned to havea well-defined configuration with the plane of the membrane beingperpendicular to the sides of the electrodes. The following areadvantageous conditions in which probing of the membrane is carried out:

1. The membrane should span the entire containment volume as exact aspossible with the plane of the membrane laying perpendicular to thesides of the electrodes.

2. The insertion of any constituents of the membrane such as membraneproteins is possible at any stage of probing.

3. It is possible to bring extracellular and inner cellular materialsinto contact with each side of the membrane.

4. The surface of the membrane is advantageously accessible for probingdevices such as a tip of the Atomic Force Microscope (“AFM”), a beam oflight, a laser of the confocal microscope, and an X-ray beam, but notlimited to these tools.

The conductive walls are fabricated on an electrically insulative base(e.g., made of silicon wafer) and can be parallel to each other andperpendicular to the base. In view of the nanometer scale height of theconductive walls and the wafer they are fabricated on, this can becalled a nanochip. Non-conductive walls surrounding the conducting wallscan be fabricated on the chip base. Two electrically conductive padsextending from each of the conductive walls are fabricated on the chipbase. The electrically conducting walls and pads can be coated with athin non-conductive layer. A membrane is disposed between the conductivewalls on the base. An electric field is propagated between theconductive walls in the plane of the membrane. The electricallyinsulative base has one or more through holes between the electricallyconducting walls where the membrane is disposed.

The chip is placed inside a container (e.g., made of silicon basedwafer) and base and can have surrounding walls. A well is formed (e.g.,by engraving) near the center of the container base directly below thehole or holes of the electrically insulative chip base when the chip isplaced inside the container. At least one passageway is formed (e.g., byengraving) in the container base and extends from the well to theoutside. A conduit may extend in each passageway. A third electrode isfabricated on the bottom of the well. An electrically conductive padextending from the electrode in the well to the wall is fabricated onthe container base.

This invention relates to probing structural changes of the membrane andthe functions associated with them as an in-plane transverse electricfield is applied between the conductive walls of the chip, usingelectric current measurements, impedance gain phase analysis, rasterscanning by atomic force microscope (AFM), and observation withconfocal, fluorescence or other microscopes, x-ray and nuclear magneticresonance (NMR), but not limited to these tools.

The invention enables probing of cellular processes such as structuralchanges of proteins, channeling mechanisms, receptor binding mechanisms,protein-protein interactions and interaction of proteins with othermolecules, in an environment that mimics an actual living cell membrane.The well represents an interior of a biological cell, and may includevarious chemical compounds, ions, proteins, RNA, DNA, organelles andvarious cellular components in a suitable medium while extracellularmaterials such as ions, ligands and other molecules in a suitable mediumcan be placed in contact with the top of the membrane. Movement of theelectrically charged particles through the membrane and electricalcharacteristics of individual proteins can be monitored by probing thevoltage change across the third electrode and another electrode, forexample the electrical conducting tip of an AFM, placed in contact withthe exterior fluid, the membrane and individual proteins therein. Theconduits extending into the well are used to introduce substances intoand out of the well.

The invention can be constructed to facilitate probing multiple cellmembranes. More than one pair of electrical conductive walls can befabricated side-by-side on the electrical insulative base with one ormore through holes between the electrical conductive walls for each ofthem. These pairs can be separated from each other by electricallynonconductive walls so that different extracellular materials and othermolecules can be placed in each of them. These pairs can be electricallyconnected in series or parallel so that the membranes can be subjectedto the same or different voltages (e.g., all cell membranes can receivethe same transverse waveforms at the same time in case of parallelconnection). The container can have multiple wells isolated from eachother accessible by individual through holes in the chip base or asingle well accessible by multiple through holes of the chip base. Thisaspect of the invention permits the use of one or more cell membrane ascontrols while the other identical cell membranes are subjected tovarious experimental conditions.

The embodiments of the invention include a combination of a chip andcontainer for the chip, an apparatus and a method for probingconformational changes of a membrane and the functions associated withthem by applying a transverse electric field in the plane of themembrane. Referring to the first embodiment, a chip includes a base.Electrically conductive walls can be spaced apart from each other andfabricated on the base. The base and the conductive walls form acontainment region that is configured to receive a membrane. Themembrane has opposing surfaces. An interior plane is bounded by themembrane surfaces and extends along the membrane. The base is comprisedof an electrically insulative material in the containment region adaptedto support at least one of the membrane surfaces. One or more throughholes are made in the non-conductive base in the area between theconductive walls preferably near the middle of the containment region.The conductive walls extend to a height above the base that is at leastas large as a thickness of the membrane and, in particular, approximatesthe membrane thickness.

One way of constructing the inventive chip is to use a flat, thin waferof a silicon-based electrically insulative material as the base. Thisbase can be flat across the entire side where the conductive walls arelocated. On the other hand, the base can be designed to be flat in thecontainment region and to have other contours outside of the containmentregion. One particularly suitable design is to fabricate the conductivewalls directly on the flat wafer. One or more through holes are made inthe wafer in the area between the conductive walls preferably near themiddle of the containment region.

The container that receives the chip can include at least twosurrounding walls and has a base made of an electrically insulativematerial. A well is formed such as by engraving near the center of thecontainer base. This well is positioned below the membrane when the chipis placed in the container. An electrode can be fabricated on thesurface of the bottom of the well and does not extend the depth of thewell. An electrically conducting pad can be fabricated on the base ofthe container and extends from the electrode in the well onto anadjacent container wall. At least one conduit is disposed in thecontainer and extends from the well through the base to the outside.

One way of constructing the container is to use a flat, thin wafer of asilicon-based electrically insulative material as the base and thewalls. The container base is fabricated such that one or more wells arefabricated in the base. This base can be flat across the entire side. Onthe other hand, the base can be designed to be flat outside and insidethe wells. An electrode flat and not extending the depth of the well isfabricated on the surface of the bottom of the well. An electricallyconducting pad extending from the electrode in the well is fabricated onthe base of the container. Two conduits extending from the well throughthe base to the outside is fabricated in the container.

Referring to the second embodiment an apparatus comprises the chip andcontainer described above, an electrical power source for applying anelectric field between the conductive walls, and an instrument adaptedto probe or image the membrane. The apparatus is adapted to apply anelectric field between the conductive walls along the plane of themembrane.

The term “in-plane electric field” is defined herein to mean an electricfield in a direction along a plane in the interior of the membrane thatextends along (i.e., generally parallel to) the membrane surfaces.Reference in this disclosure to applying the electric field along theplane of the membrane may not require the electric field direction to beexactly parallel to the surfaces of the membrane. In the case where themembrane is not precisely positioned flat on the base, the electricfield may not be propagated exactly parallel to the membrane surfacesand yet this is sufficient for purposes of the invention. It will beapparent to one of ordinary skill in the art that the invention needonly refer to one membrane plane as a point of reference to accuratelydescribe application of the in-plane electric field. Reference to theconductive walls having a height approximating a thickness of themembrane means spanning the membrane thickness.

The third embodiment of the invention features a method for probingstructural changes in a membrane and functions associated with them byapplying an electric field. The chip and container described above areprovided, a membrane is positioned between the conductive walls incontact with the chip base and traversing the through hole; a firstmaterial is added on top of the cell membrane; a second material isinserted through the passageway into the well; and an electric field isapplied between the conductive walls along the plane of the membrane.

In view of the use of the present invention with biological cellmembranes and biological cells, the chip and the container can besterilized, dried with an inert gas, and packaged in a sterilecondition. Each chip is disposable after being used for one membranewhile the container can be reused.

Specific features of the invention will now be described. The apparatusfor imaging or probing the membrane includes an instrument selected fromthe group consisting of an atomic force microscope, a confocalmicroscope, a confocal laser scanning microscope, a fluorescencemicroscope, X-ray spectrometer, NMR impedance gain phase analyzer, andcombinations thereof. The apparatus includes an instrument adapted todetermine the properties of the membrane, including topographical,electrical, viscous, and elastic properties.

As defined herein the membrane can be a bilayer comprised of amphiphiliclipids, cholesterols, proteins, a part of a cell membrane, andcombinations thereof. The membrane can include constituents selectedfrom the group consisting of glycolipids, phospholipids, cholesterol,proteins, and combinations thereof.

In particular, the height of the conductive walls approximates thethickness of the membrane, e.g., the monolayer, bilayer or cells asdescribed above. The invention contemplates using conductive wallshaving heights greater than the membrane thickness so long as this doesnot significantly interfere with application of the in-plane electricfield. In particular, the membrane can extend to a height of not morethan 10 nanometers from the base, specifically, ranging from 5 to 10nanometers. The spacing between the conductive walls is selected to besufficient to accommodate the membrane sample being probed and use of anelectric field of an appropriate magnitude and in particular, can be notmore than 10 micrometers and in particular from 3 to 10 micrometers. Thechip base and its conductive walls can be configured whereby the wallsextend perpendicular to the base and parallel to each other. The basecan be flat in a containment region located between the conductive wallswhere the membrane is disposed. In one aspect of the invention there isno cover over the containment region. This enables insertion of membraneconstituents and other substances or articles, which can be used orwhose effect on structural transitions can be probed by application ofthe in-plane electric field.

The topmost surface of the membrane located in the containment regioncan be contacted with an electrode such as a tip of an Atomic ForceMicroscope (“AFM”), forming an electric field between the tip and thethird electrode in the well. This advantageously permits applying afield to individual molecules in the membrane, such as to an individualprotein, perpendicular between the tip and the third conductor. The tipof the AFM can be moved to apply the electric field one at a time to aplurality of proteins in contact with the membrane.

The voltage source used in the apparatus is adapted to apply direct oralternating voltage along the plane of the membrane. The voltage sourcecan apply voltage pulses in the form of square or triangular pulses,sine wave, or other form of pulses. An electrical resistor can beconnected to an electrical lead extending from the voltage source and toone of the conductive pads of the conductive walls. Another electricallead from the other conductive wall is connected to the voltage source.A voltage drop across the resistor is monitored using an oscilloscopewhile varying the electric field applied along the plane of themembrane.

Referring to specific aspects of the method, in one technique the cellmembrane can be contacted with a protein (e.g., the protein can beinserted in the membrane). The electric field is applied along the planeof the cell membrane and the effect of the electric field on the proteinis monitored. Proteins that span the membrane, such as transmembraneproteins, proteins that have residues extending in the membrane, orproteins that are bound or interact with other proteins or moleculesthat contact the membrane, can be investigated using the presentinvention. For example, the investigated protein can contact at leastone molecule, and that molecule can directly contact the cell membrane.Alternatively, that molecule can contact another molecule that directlycontacts the cell membrane. The method includes monitoring an effect ofthe electric field on a constituent of the cell membrane selected fromthe group consisting of a receptor protein, channel proteins andcombinations thereof.

In one technique the cell membrane can be contacted with a protein(e.g., the protein can be inserted in the membrane). The electric fieldis applied along the plane of the cell membrane and the effect of theelectric field on the protein is monitored. Transportation of elementssuch as ions and small molecules from one side of the membrane to theother side can be investigated by using the present invention. Thebinding of proteins and other molecules to the transmembrane proteinscan be investigated by the present invention.

Various techniques may be used to position the membrane in thecontainment region. It is desirable to achieve precise alignment of thelower membrane surface on the base and the ends of the membrane incontact with the conductive walls. In one technique, the membrane ispositioned on the base in the containment region by filling liquid inthe containment region, forming the membrane on the surface of theliquid, and subsequently evaporating the liquid causing the membrane todispose on the base in the containment region over the through hole orholes. In another technique the membrane is positioned on the base inthe containment region by immersing the chip in a liquid, forming themembrane on the surface of the liquid, lifting the base from the liquidcausing the liquid to leave the containment region, and causing themembrane to depose on the base in the containment region over thethrough hole or holes.

The present invention offers numerous advantages. The invention enablesprobing of membranes by applying the transverse in-plane electric field,which heretofore was not possible. The invention permits investigatingmembrane systems on the scale of a monolayer or bilayer, which willuncover behavior that may not be observable in bulk. One particularlyvaluable aspect of the invention is its ability to analyze the cellmembrane. Various cell membranes can be used from various cell andtissue types, conditions and environments. This can be done with extremeversatility. The membrane can be formed so that its composition isknown. The specific lipids can be extracted and purified from existingmembranes and used in the sample membrane. Specific proteins can beinserted one at a time at particular locations in the membrane. Theseproteins can be inserted at isolated locations or at locations nearother proteins to enable probing of their interaction. On the otherhand, the invention offers the flexibility of designing the samplemembrane with a plurality of proteins. The membrane can be made toinclude a variety of different proteins and other membrane constituents.The sample membrane can be a portion of an actual biological cellmembrane.

The membrane can be probed in a variety of ways. Because the containmentregion can be without a cover, the membrane can be physically contactedbefore, during and after application of the in-plane electric field. Forexample, the tip of an atomic force microscope can be scanned over theexposed outermost membrane surface in the containment region. Microscopyof the membrane can be carried out, for example, using one or more ofthese, Atomic Force, Laser Scanning Confocal, and Fluorescentmicroscopes. The invention enables a thorough evaluation of the variousproperties of the membrane, including topographical, electrical,viscous, and elastic properties. The membrane also can be positioned sothat it can be accessed from one or two sides of the containment region.This offers additional probing, for example, using X-ray spectroscopy.

Not only can changes in the membrane be probed by applying the electricfield, but how the constituents of the membrane interact with othermolecules and external stimuli can be probed. For example, the membranecan be made to come in contact with including but not limited to: drugsor therapeutics, chemicals, ligands, fluorescence tags, siRNA, antisenseRNA, hormones, enzymes, antibodies, viruses, and combinations thereof.For example, one can reconstruct and probe cancer cells and monitortheir response to certain molecules of interest, such as anticancerdrugs, with and without application of the in-plane electric field.Similarly, one can investigate the capillary endothelial cells of theblood brain barrier and response to certain therapeutics uponmanipulation of carriers by applying the in-plane electric field and/orthe perpendicular field between the tip of the atomic force microscopeand the third conductor. The treatment of the cells can be carried outbefore, during or after probing of the membrane by application of thein-plane electric field.

The invention can be used to probe the cellular response to externalstimuli such as light. The invention can be used to investigate cellulartransport. It may be possible to manipulate a protein channel of amembrane in the containment region, to change its conformation from anopen conformation to closed conformation. The invention can be used toinvestigate ligand-receptor binding processes. The binding sites can beidentified by scanning the receptor protein with AFM before, during andafter a ligand binding to a receptor protein. and the rate of ligandbinding can be determined.

The sample membrane can be subject to various treatments and combinedwith various probing techniques to investigate properties including, butnot limited to electric, viscous, elastic, and topographical properties.For example, certain proteins of interest can be fluorescently taggedand then monitored using a fluorescent microscope, while observing theirtopographical structural transitions using AFM, in response toapplication of the in-plane electric field. Impedance of the membraneand the electrical characteristics of the membrane can be analyzed todetermine the electric, viscous, elastic properties associated withconformational transitions.

It is advantageous for a chip to include a plurality of containmentregions isolated from each other by surrounding walls so that multipleexperiments can be conducted simultaneously using the chip. Thecontainment regions can be electrically addressed with the in-planeelectric field all at the same time by electrically interconnecting theconductive walls of the chip. On the other hand, the chip can befabricated in a way that enables the containment regions to beelectrically independently addressed with the in-plane electric field atdifferent times and different electric fields. One of ordinary skill inthe art will recognize that these are but a few examples of innumerableapplications of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Illustrates a configuration of the inventive chip in across-sectional side view as seen along the plane 1-1 in FIG. 2;

FIG. 2: Illustrates a top view of the chip of FIG. 1;

FIG. 3 illustrates a membrane probed in the present invention;

FIG. 4: Illustrates a configuration of the inventive container for thechip in a top view;

FIG. 5 a,b: Illustrate cross-sectional side views of the inventivecontainer rotated 90 degrees; FIG. 5 a being a view as seen along theplane 5 a-5 a in FIG. 4; FIG. 5 b being a view as seen along the plane 5b-5 b in FIG. 4; and FIG. 5 a shows the alignment of the inventive chipbefore being inserted into the container;

FIG. 6: Illustrates a top view of the inventive device including thechip received in the container;

FIG. 7 a, b: Illustrate cross-sectional side views of the device of FIG.6 rotated 90 degrees, FIG. 7 a being a view as seen along the plane 7a-7 a in FIG. 6; FIG. 7 b being a view as seen along the plane 7 b-7 bin FIG. 6; FIG. 7 a being without the third electrical conductor orconducting pads on the container for clarity; and

FIG. 8: Illustrates a cross-sectional side view of the inventive devicesimilar to the view shown in FIG. 7 a, with membrane and application offirst and second voltage sources to the membrane.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, an apparatus 10 including a device 12including a chip 14 and a container 16 into which the chip is received.The chip has an electrically insulating thin wafer as a base 18 on whichfirst and second nano-meter scale electrically conducting walls 20, 22are fabricated. The wafer has a flat upper or working surface 24. Theconductive walls extend perpendicular to the base and are spaced apartapproximately parallel to each other by a distance D1. A containmentregion or volume 26 has a width defined between the conductive walls atdistance D1, a height D2 (FIG. 5) defined between the surface of thewafer and upper surfaces of the conductive walls and a length D3 definedby a length of the conductive walls. Electrically conducting pads 28, 30extending from the electrically conducting walls 20, 22, respectively,are also fabricated on the wafer and can extend to a periphery thereof32. The height of the pads may be smaller or larger than the height ofthe conductive walls.

Referring to FIG. 3, a membrane 40 is disposed in the containmentregion. The membrane composed of a bilayer has opposing spacedapart-surfaces 42, 44 which extend across a major area of the membrane,an interior 46 between these surfaces and a thickness t. An interiorplane P of the membrane is bounded by the membrane surfaces and extendsalong the membrane (i.e., approximately parallel to the membranesurfaces). The membrane can comprise a monolayer 48 a or 48 b havingspaced apart surfaces 42 or 44 and surfaces 43 or 45, an interiorbetween these surfaces and a thickness t_(a) or t_(b). The interiorplane P of the monolayer would extend between the spaced apart surfaces.

It should be appreciated that certain membranes, as in the case of thebiological cell membrane, have an inner membrane surface and opposing,spaced-apart outer membrane surfaces, which may have different localizedcompositions and concentrations of proteins and other constituents, iongradients and the like compared to the other membrane surface. Themembrane can comprise a monolayer 48 a or 48 b or bilayer 50 of naturalamphiphilic lipids 52 found in the biological cell membrane includingglycolipids, phospholipids, synthetic amphiphilic lipids, otherconstituents of the biological cell membrane including cholesterol andproteins (e.g., a channel protein 54 or a receptor protein), and anycombination of them. Moreover, any of these constituents can be addedany time after the initial membrane is formed in the containment region.The invention is suitable for probing membranes of any chemicalcomposition, even non-biological membranes. A portion of a biologicalcell membrane can be deposited in the containment region. An entirebiological cell or cells may be deposited in the containment region,which forms the membrane as that term is used in this disclosure. Inthis case, the electrical conducting walls may be sized and configuredto appropriately accommodate the entire cell or cells in the containmentregion.

The bilayer can be formed by extracting and purifying amphiphilic lipidsfrom biological cell membranes and using these lipids to form thebilayer. Other cell membrane constituents, such as cholesterol andproteins, can be extracted and inserted one or more molecule at a timeinto the lipid bilayer. The membrane constituents can be inserted intothe bilayer before or after application of the electric field. Forexample, in the case of probing the structural changes of a particularprotein of interest (e.g., a channel protein 54 or a receptor protein)one or a limited number of proteins can be inserted and then theirresponse to the in-plane field can be monitored. On the other hand, themembrane can be formed with many proteins, and then the proteins can bemonitored in response to the in-plane electric field. The bilayer can beformed by combining one or more chemically or enzymatically synthesizedmolecules (e.g., lipids).

The electrically conductive walls 20, 22 are fabricated on the chip baseor wafer 18 and extend upwardly to a height D2 at least as high as themembrane thickness t, ta or tb in the containment region (i.e., thedistance between the membrane surfaces in a direction perpendicular tothe base). In particular, the conductive walls can have a height D2 thatapproximates the thickness t, t_(a), or t_(b) of the membrane (FIG. 5),for example, a height in a range of 5 to 10 nanometers from the surfaceof the conductive wafer. The membrane can have a height D2 approximatingthe thickness of a molecular monolayer or bilayer and, in particular,the thickness of a biological cell membrane, or biological cell or cellswhen positioned in the containment region. The distance D1 between theconductive walls can vary as desired depending on the sample. Forexample, the distance is in a range of about 3 to 10 micrometers. Thechip includes at least one through hole 60 that can be centered betweenthe conductive walls in the containment region. The membrane 40 deposedin the containment region extends over the through hole.

The containment region 26 advantageously is exposed or uncovered on thetop and in particular, on one or two sides 62, 64 as well. An advantageof the invention is that it enables probing of the top surface of themembrane and, optionally, sides of the membrane. In addition, the opentop of the containment region permits adding any component to themembrane before, during and after application of an electric field,including but not limited to membrane constituents. When the membrane 40is positioned in the containment region, the lowermost membrane surface44 is in contact with surface 24 of the wafer and covers the opening 60while the opposing topmost membrane surface is exposed to probing. Thewafer provides stability of the membrane in the containment region whilethe portion of the membrane extending over the opening permits passageof material upwards and downwards into or through the membrane. Twosides of the membrane (e.g., sides 56, 58) are in contact with theconductive walls 20, 22. One or both of the other sides of the membrane66, 68 can also be exposed to probing near open sides of the containmentregion 62, 64 of the chip.

Referring to FIGS. 4 and 5, the container 16 includes an electricallyinsulative base 70 and wall 72 extending around the periphery thereof.The wall 72 can completely surround the container base or can extend ononly two opposite sides thereof. First and second conducting pads 71, 73are disposed on the container walls in alignment with the pads 28, 30.

The container base includes a well 74 centered beneath the through hole60 and the containment region of the nanochip. A third electrode 76 isfabricated in between a plane P1 of the inner surface of the firstconducting wall and a plane P2 of the surface of the second conductingwall (FIG. 5), on the floor 78 of the well beneath the containmentregion and on a side wall 80 of the well. The third electrode 76 isspaced from the conductive walls so as to be electrically isolatedtherefrom. Electrical conducting pad 82 is fabricated on the containerwall, and on the container base into contact with the third electrode76. Ingress and egress passageways 84, 86 extend from an exteriorsurface of the container to the well 74. Each passageway includes aconduit or needle 88, 90 disposed therein.

The bottom of the chip base 92 contacts the upper base surface 94 of thecontainer (FIG. 5). The perimeter of the chip base 32 contacts theinside surface 96 of the container walls. This contact between chip andcontainer establishes an electrical connection between the conductivepads 28, 30 of the chip and electrodes 71, 73, respectively, fabricatedin alignment with the pads on the container wall 72.

The first conducting wall, second conducting wall, pads on the chip,third electrode and pads on the container and container wall, can bemade of any electrical conducting material, for example, Au, Ag or Cubut not limited to these. The wafer and container can be made of nonelectrical conducting materials, for example, Si/SiO₂ but not limited tothese.

Various extracellular or outer components, natural or synthetic, in asuitable medium, or other components that would be apparent to oneskilled in the art in view of this disclosure, (77) can be disposed ontop of the membrane when it extends over the through hole (FIG. 8).Various inner cellular or inner components, natural or synthetic, in asuitable medium, or other components that would be apparent to oneskilled in the art in view of this disclosure, (79) can be fed into thewell though ingress 84 and can be extracted from the well through aegress 86. Examples of suitable extracellular components are ions andligands. Examples of suitable inner cellular components are componentsselected from the group consisting of RNA, DNA, proteins, ions, cellorganelles and combinations thereof. Suitable mediums include water, andvarious natural or synthetic buffers. The membrane components and innerand outer components and mediums may establish any ion gradients acrossthe membrane that are desired, such as those exhibited by certainmembranes in their particular biological environment.

Referring to FIGS. 1 and 3, a first voltage source applies an electricfield E1 along the plane P of the membrane 40 when it is positioned sothat the bottom surface 44 is disposed on the wafer and the membranessides 56, 58 contact inner surfaces 98, 100 of the conductive walls. Itis desirable to position the membrane flat on the wafer. This assists inprobing the exposed top surface 42 of the membrane at a known location.In addition, as the conductive walls are perpendicular to the wafer, theelectric field E1 can be applied along the plane of the membrane P.Electrical leads 102, 104 from a voltage source 106 (FIG. 8) areconnected to the electrical conducting pads 28, 30, respectively,enabling application of a voltage to the conductive walls thatpropagates electric field E1 between the electrically conducting wallsin a direction that is perpendicular to them along the plane P of themembrane when the lower membrane surface is disposed flat on the waferin the containment region. Alternatively, if the conductive pads 28, 30extend to the periphery of the chip as in FIG. 7 a and conducting pads71, 73 are fabricated on the wall of the container, making an electricalconnection therebetween, the voltage can be applied by leads 102, 104 tothe conductors 71, 73 on the wall of the container to propagate theelectric field E1 along the plane of the membrane.

A second voltage source 108 can apply a voltage to the third electrodeor not. An electric field E2 may thus be established in the membranebetween an electrode 110 such as the tip of the atomic force microscopein contrast with the membrane (represented by an arrow in FIG. 8) andthe third electrode. This electric field E2 may extend in a directionperpendicular to the base.

The first and second voltage sources may apply a direct or alternatingvoltage parallel to or perpendicular to the plane P of the membrane,respectively. The voltage sources can apply voltage pulses, for example,square pulses, triangular pulses, and sine wave. The amplitude and thefrequency of the voltage pulse can be selected by one of ordinary skillin the art in view of this disclosure. The voltages will be applied atsuitable magnitudes for causing structural changes to the constituentsof the membrane.

Structural transitions in the membrane and the proteins embedded in orin contact with the membrane are caused by the reorientation of themembrane molecules coupled to the in-plane electric field. Structuralchanges to the membrane in response to application of the in-planeelectric field are probed by electric current measurements, atomic forcemicroscope, confocal microscope, fluorescent microscope and X-rayspectrometer, but not limited to these tools. Viscous, elastic andelectric properties of the membrane can be measured in accordance withthe invention.

One particularly suitable instrument of the inventive apparatus forprobing the bilayer is an Atomic Force Microscope 112 (“AFM”) (FIG. 8),which is capable of detecting structural changes taking place in theconstituents of the membrane. The atomic force microscope and otherspectroscopic instruments are able to determine the structure of themembrane through the open top or sides before, during, and after anelectrical field is applied in the device. Of course, while the opensurfaces of the containment region is an advantageous feature of thepresent invention, one of ordinary skill in the art will appreciate inview of this disclosure that a cover for the open top or sides of thecontainment region is desirable in some instances. For example, a cover(e.g., glass or plastic coverslip) might be placed on top of theconductive walls before, after, and in some cases during, microscopy toprevent any contamination or to prevent drying of the membrane. Certainconstituents of the membrane and the cells may be fluorescently labeledand can be viewed through a cover using a fluorescent microscope whilean electric field is applied in the device.

The inventive chip can include one or more containment regions 26. Thefirst voltage source 106 can electrically address the conductive wallsof each containment region with the same or different voltage magnitudesand times. In addition, the voltage source 106 can apply voltage tocontainment regions on different chips at the same time. The membranes40 that are positioned in the multiple containment regions can be thesame or different, for example, some containment regions of a chipincluding bilayers or monolayers, other containment regions of the chipincluding cells, and still other containment regions of the chipincluding cell membranes, or all containment regions of the chipincluding cell membranes but from different cell or tissue types orsubject to different treatments. Each container can have one or morewells that each communicates with numerous through holes in a chip orwells that are each isolated from each other and each communicate onlywith one through hole of the chip.

Many modifications and variations of the invention will be apparent tothose of ordinary skill in the art in light of the foregoing disclosure.Therefore, it is to be understood that, within the scope of the appendedclaims, the invention can be practiced otherwise than has beenspecifically shown and described.

1. A method for probing changes of a membrane by applying an electricfield, comprising: providing a device including a base comprised ofinsulating material, electrically conductive walls spaced apart fromeach other and disposed on said base, wherein said conductive walls areapproximately perpendicular to said base and parallel to each other,wherein said conductive walls and said base are configured to receive amembrane selected from the group consisting of a monolayer or bilayercomprised of amphipathic lipids, a cell membrane comprised of lipids,and combinations thereof, said membrane having opposing top and bottommembrane surfaces and an interior reference plane that is bounded bysaid membrane surfaces and extends along said membrane, wherein saidconductive walls each has a conductive side that extends to a heightabove said base that is at least as large as a thickness of saidmembrane, and a through hole formed in said base between said conductivewalls; providing an enclosure below said base including a well belowsaid through hole; providing a passageway extending from a periphery ofsaid enclosure to said well; positioning said membrane between saidconductive walls such that said bottom surface of said membrane contactssaid base and extends over said through hole, wherein said membrane hasside surfaces which are in contact with said conductive walls; adding afirst material on top of said membrane; adding a second material throughsaid passageway into said well; and applying an alternating electricfield between said conductive walls along the plane of said membranethat causes changes in said membrane including reorienting molecules ofsaid membrane; and monitoring said changes in said membrane caused byapplication of said electric field.
 2. The method of claim 1 whereinsaid reorientation of said molecules of said membrane cause structuraltransitions of a protein in contact with said membrane, comprisingmonitoring said structural transitions of said protein.
 3. The method ofclaim 1 comprising insulating material disposed on said conductivewalls.
 4. A method for probing changes of a membrane by applying anelectric field, comprising: providing a device including a basecomprised of insulating material, electrically conductive walls spacedapart from each other and disposed on said base, wherein said conductivewalls are approximately perpendicular to said base and parallel to eachother, wherein said conductive walls and said base are configured toreceive a membrane selected from the group consisting of: a monolayer orbilayer comprised of amphipathic lipids, a cell membrane, a molecularbilayer, a molecular monolayer, a non-biological membrane, andcombinations thereof, said membrane having opposing top and bottommembrane surfaces and an interior reference plane that is bounded bysaid membrane surfaces and extends along said membrane, wherein saidconductive walls each has a conductive side that extends to a heightabove said base that is not more than 10 nanometers, and a through holeformed in said base between said conductive walls; providing anenclosure below said base including a well below said through hole;providing a passageway extending from a periphery of said enclosure tosaid well; positioning said membrane between said conductive walls suchthat said bottom surface of said membrane contacts said base and extendsover said through hole, wherein said membrane has side surfaces whichare in contact with said conductive walls; adding a first material ontop of said membrane; adding a second material through said passagewayinto said well; applying an electric field between said conductive wallsalong the plane of said membrane; and monitoring changes in saidmembrane caused by application of said electric field.
 5. A method ofclaim 4 comprising: providing electrically conducting pads extendingfrom said conductive walls toward a periphery of said base and providinga container as said enclosure, said container being comprised ofinsulating material.
 6. The method of claim 4 wherein said firstmaterial is selected from the group consisting of ions, ligands andcombinations thereof.
 7. The method of claim 4 wherein said secondmaterial comprises components selected from the group consisting of RNA,DNA, proteins, ions, cell organelles and combinations thereof.
 8. Themethod of claim 4 comprising monitoring an effect of said electric fieldon a protein in contact with said membrane.
 9. The method of claim 8wherein said protein is selected from the group consisting of a receptorprotein, a membrane transport protein, a transmembrane protein, an ionchannel and combinations thereof.
 10. The method of claim 4 comprisingprobing or imaging said membrane using an atomic force microscope,comprising contacting said membrane with an electrically conductive tipof said atomic force microscope, providing a third electrode on a lowersurface of said well and establishing electrical conduction between saidconductive tip and the said third electrode.
 11. The method of claim 4comprising probing or imaging said membrane using an instrument selectedfrom the group consisting of: atomic force microscope, confocalmicroscope, laser scanning confocal microscope, scanning electronmicroscope, transmission electron microscope, phase contrast microscope,differential-interference-contrast microscope, dark-field microscope,bright-field microscope, electron microscope tomography, fluorescencemicroscope, X-ray spectrometer, impedance gain phase analyzer andcombinations thereof.
 12. The method of claim 4 comprising measuringproperties of said membrane selected from the group consisting of:topographical, electrical, viscous, elastic, and combinations thereof.13. The method of claim 4 comprising applying said electric field tosaid membrane as voltage pulses.
 14. The method of claim 13 wherein saidvoltage pulses are selected from the group consisting of square pulses,triangular pulses, and sine wave.
 15. The method of claim 4 wherein saidmembrane includes constituents selected from the group consisting ofglycolipids, phospholipids, cholesterol, proteins, and combinationsthereof.
 16. The method of claim 4 comprising inserting a protein intosaid membrane, applying said electric field along the plane of saidmembrane and using said microscopy to monitor the effect of saidelectric field on said protein.
 17. The method of claim 4 comprisingcontacting a surface of said membrane with a tip of an atomic forcemicroscope.
 18. The method of claim 4 comprising treating said membraneand observing a response to said treatment.
 19. The method of claim 18wherein said treatment is selected from the group consisting of: drug,therapeutic, chemical, enzymatic, ligand, fluorescence, siRNA, antisenseRNA, hormonal, antibody, viral, bacterial and parasitic treatments, andcombinations thereof.
 20. The method of claim 4 comprising usingmicroscopy to monitor an effect of said electric field on a protein ofsaid membrane.
 21. The method of claim 4 comprising insulating materialdisposed on said conductive walls.